Single Nucleus MultiOmics and In Vivo Massively Parallel Reporter Assays Identify Functional Sex-Biased Enhancers Linked to Liver Metabolism and Disease

Growth hormone (GH) controls sexual dimorphism in hepatocyte gene expression programs governing lipid metabolism, bile acid synthesis and xenobiotic processing, which contribute to sex differences in metabolic dysfunction-associated steatotic liver disease (MASLD) risk. Despite extensive study of GH-regulated sex differences in gene transcription, the functional cis-regulatory hepatocyte enhancers that orchestrate these sex-dependent metabolic programs remain largely unknown. Here, we integrated single-nucleus multiomic profiling of hepatocyte chromatin accessibility with in vivo functional enhancer assays to identify and validate GH-responsive, sex-biased hepatocyte enhancers in intact mouse liver. We constructed a tiled HDI-STARR-seq library of 23,912 reporters spanning 1,839 liver ATAC regions and delivered it to liver by hydrodynamic injection, enabling functional assessment of enhancer activity in vivo across distinct biological conditions. Reporters representing 840 ATAC regions showed sex-biased and/or GH-regulated enhancer activity, in many cases mirroring the regulation of their chromatin accessibility in hepatocytes, validating these sites as functional, physiologically regulated enhancers. The regulated enhancers were enriched for activating histone marks (H3K4me1, H3K27ac), for binding sites for the GH-activated transcriptional regulator STAT5, and for the STAT5-dependent, sex-specific repressors BCL6 and CUX2. Further, de novo motif analysis identified binding sites for HNF4A and for several novel factors specifically enriched at the regulated enhancers. Sex-biased and GH-regulated functional enhancers were linked to both MASLD-enabling and MASLD-protective genes, suggesting that GH-dependent chromatin remodeling at these loci contributes to sex-differential metabolic disease susceptibility. This integrated in vivo approach defines a validated set of GH-regulated hepatocyte enhancers through which chromatin accessibility and transcription factor binding drive sexual dimorphism in hepatic metabolism and sex-specific MASLD risk.